During gram-negative sepsis monocytes/macrophages (MO) respond to bacterial endotoxin (LPS) by releasing cytokines. While great progress has been made in defining the mechanism of action of these cytokines there is a significant gap in our knowledge about MO receptors and second messenger pathways relaying signals from LPS. Recent discoveries from this laboratory have provided new insights into events at the plasma membrane surface that control LPS recognition. In blood, LPS forms a high affinity complex with a plasma protein, LPS binding protein (LBP). This complex binds to the MO plasma membrane via a specific receptor we have identified as CD14. Engagement of CD14 by LPS-LBP complexes is followed by the rapid production of MO cytokines such as TNF or IL-1. CD14 is linked to the cell membrane by a glycosol-phosphatidyl inositol linkage and thus does not have cytoplasmic and/or transmembrane domains with identifiable structures, e.g. tyrosine kinase, G-protein linkage, etc. Furthermore, cytokine production from MO is not mimicked by Ca 2+ ionophores and/or phorbol esters. Therefore, we hypothesize that LPS signals via the LBP/CD14-dependent pathway are relayed by kinases and/or protein phosphatases that may be associated directly with or linked to the LBP/CD14 pathway and that are activated after engagement of CD14 by LPS-LBP complexes. The consequence of this is the initiation of a cascade of intracellular phosphorylations. The experiments described in this application are designed to systematically characterize the role of phosphoprotein intermediates involved in relaying information from LPS via the LBP/CD14 pathway.